Method and apparatus for amplifying nucleic acids

ABSTRACT

Provided are a method and apparatus for amplifying nucleic acids. The method includes introducing into a reaction vessel via different inlet channels a reactant aqueous solution containing reactants for nucleic acid amplification and a fluid that is phase-separated from the reactant aqueous solution and does not participate in amplification reaction, creating a plurality of reactant aqueous solution droplets surrounded by the fluid by contacting the reactant aqueous solution with the fluid in the reaction vessel, and amplifying the nucleic acids in the reactant aqueous solution droplets. The apparatus includes a substrate, a reaction vessel formed inside of the substrate, at least one first inlet channel formed inside the substrate, connected to an end of the reaction vessel, and allowing introduction of a reactant aqueous solution containing reactants for nucleic acid amplification into the reaction vessel, a second inlet channel formed inside the substrate, connected to the end of the reaction vessel, and allowing introduction of a fluid that is phase-separated from the reactant aqueous solution and does not participate in amplification reaction into the reaction vessel, and a heating unit installed on the substrate in such a way to thermally contact with the substrate and heating the substrate.

BACKGROUND OF THE INVENTION

This application claims the priority of Korean Patent Application No.10-2004-0016795, filed on Mar. 12, 2004, in the Korean IntellectualProperty Office, the disclosure of which is incorporated herein in itsentirety by reference.

1. Field of the Invention

The present invention relates to a method and apparatus for amplifyingnucleic acids, and more particularly, to a method and apparatus foramplifying trace amounts of nucleic acids to a level necessary for aspecific analysis.

2. Description of the Related Art

To disclose the genetic information of nucleic acids such as DNAs andRNAs for the purpose of sequence analysis and disease diagnosis,amplification of trace amounts of nucleic acids to a desired level isrequired. As nucleic acid amplification techniques well known inordinary persons skilled in the art, there are a typical isothermalamplification technique, such as ligase chain reaction (LCR),strand-displacement amplification (SDA), nucleic acid sequence-basedamplification (NASBA), transcription-mediated amplification (TMA), andloop-mediated isothermal amplification (LAMP), and a non-isothermalamplification technique, such as polymerase chain reaction (PCR) thathas been recently mainly used.

Among the amplification techniques, the PCR is performed by repeatedcycles of three steps: denaturation, annealing, and extension. In thedenaturation step, a double-stranded DNA is separated into two singlestrands by heating at 90° C. or more. In the annealing step, two primersare each bound to the complementary opposite strands at an annealingtemperature of 55 to 65° C. for 30 seconds to several minutes inconventional PCR machines. In the extension step, DNA polymeraseinitiates extension at the ends of the hybridized primers to obtain DNAdouble strands. The time required for the extension step variesdepending on the concentration of a template DNA, the size of anamplification fragment, and an extension temperature. In the case ofusing common Thermusaquaticus (Taq) polymerase, the primer extension isperformed at 72° C. for 30 seconds to several minutes.

However, with respect to nucleic acid amplification according to theabove-described PCR technique, when nucleic acids are present in traceamounts, PCR efficiency may be lowered. For this reason, there arises aproblem in that amplification is hardly performed or nonspecific PCRproducts are often produced.

To solve this problem, Kemp et al. suggested a Nested PCR technique inwhich a two-step PCR is performed using an outer primer pair and aninner primer pair (David J. Kemp et al., 1989, Proc. Natl. Acad. Sci.Vol. 86, 2423˜2427). That is, the Nested PCR technique is a techniquethat prevents production of nonspecific PCR products by performing afirst PCR using the outer primer pair and a second PCR using the innerprimer pair. However, if the outer primer pair is not removed prior toinitiating the second PCR, there may be a problem in that interactionbetween the outer primer pair and the inner primer pair must beconsidered. Furthermore, since cross contamination may occur duringopening of a reaction vessel in the interim between the first PCR andthe second PCR to insert PCR reactants for the second PCR to thereaction vessel, the Nested PCR technique requires a special attention.

Recently, there has been reported a fast PCR technique starting with asingle molecule using a micro device in which a reaction vessel is small(E. T. Lagally et al., 2001, Anal. Chem. 73, 565˜570). In this PCRtechnique, however, nucleic acids may be abnormally adsorbed on asurface of the micro reaction vessel made of silicon or glass, therebyadversely affecting PCR amplification. Furthermore, PCR reactants may beevaporated due to their small volume.

Meanwhile, when PCR temperature reaches below the melting temperature ofnucleic acids during the PCR, nonspecific PCR products such asprimer-dimers may be produced.

To solve this problem, there have been suggested a technique in whichcommon components of PCR amplification, such as DNA polymerase, are notinserted until a first cycle reaches the melting temperature of nucleicacids, and a hot start PCR technique. In detail, there are a method ofadding common PCR components after a first cycle reaches the meltingtemperature of nucleic acids, a method in which a wax bead placed on areaction solution is melted by heating to form a solidified layer havingcommon PCR components thereon so that the reaction solution is mixedwith the common PCR components after a first cycle reaches the meltingtemperature of nucleic acids, and a method using a Taq DNA polymeraseantibody in which while a first cycle reaches the melting temperature ofnucleic acids, Taq start antibody is released from Taq DNA polymerase sothat the Taq DNA polymerase is activated. However, according to theabove methods, there is a burden to add PCR reactants during PCR andcross contamination may occur. Furthermore, in the case of using Taqstart antibody, an activation time of 10 minutes or more is normallyrequired.

In PCR technique, the amount of PCR products after n cycles would betheoretically 2^(n)-fold of the initial amount of target nucleic acids.The amount of PCR products in an initial PCR step increaseslogarithmically according to the number of cycles. However, since theannealing efficiency of primers and the synthesis efficiency of DNAdouble strands are actually not 100%, when the amount of PCR productsreaches a predetermined level, there is observed a plateau effect inwhich an increase rate of PCR products decreases and amplificationfinally stops. In this regard, it is difficult to deduce the initialamount of target nucleic acids from the amount of PCR products.Conventionally, to quantify the initial amount of target nucleic acids,an internal standard sample is used.

Kopp et al. suggested a continuous-flow PCR on a chip in which a PCRsolution flows in a reaction vessel with different temperature areas viaa micro channel so that continuous PCR amplification is carried out(Martin U. Kopp et al., 1998, Science, Vol. 280, 1046˜1048). Since thisPCR technique is not based on heating the entire surfaces of thereaction vessel, the reaction rate is determined by a flow rate, not aheating/cooling rate. However, separate channels for several standardsamples are required for quantitative analysis, which increases a chipsize. Furthermore, a large number of chips for repeated experiments arerequired. Amplification of different samples using a single channel maycause a problem such as contamination by DNAs adsorbed on the surface ofthe channel.

Baker et al. suggested PCR amplification based on fluid movement on aglass chip (Jill Baker et al., 2003, Micro TAS, 1335-1338). According tothis PCR technique, the temperature of the glass chip is controlled insuch a manner that the entire surface of the glass chip is raised by aheater between channels and is cooled by a cooling water beneath theglass chip. Baker et al. reported that when a fluorescent signal wasmeasured during thermal PCR cycles in a channel filled with a dilutedsample, a fluorescent peak was detected on a single molecule. However,accomplishment of this result required an analysis procedure such asremoval of a background signal from weak fluorescent signal. Therefore,the analysis procedure of a fluorescent signal is obscure and it isimpossible to determine whether a fluorescent peak originates from asingle molecule.

Nakano et al. suggested a single molecule PCR technique usingwater-in-oil emulsion as a reactor (Michihiko Nakano et al., 2003, J.Biotechnology, 102, 117-124). In detail, first, an aqueous solutioncontaining a PCR mixture, oil, and a surfactant are placed in a PCR tubeand mixed using a magnetic stirrer bar to obtain the water-in-oilemulsion. Initial several cycles of PCR amplification are performed insmall aqueous solution droplets. When the small aqueous solutiondroplets are united by phase separation of the aqueous solution and theoil by centrifugation, later cycles of PCR amplification proceed. Thiscompletes the single molecule PCR suggested by Nakano et al. Generally,when the concentration of template DNAs is very low, hybridizationbetween primers of relatively high concentration occurs more easily,relative to that between the template DNAs and the primers, therebyproducing nonspecific PCR products. In this regard, the single moleculePCR technique uses the water-in-oil emulsion as the reactor so thatprimary PCR amplification occurs in aqueous solution droplets containinghigh concentration template DNAs. When primers, which are one ofreactants in the aqueous solution droplets containing the template DNAs,are completely consumed during initial several cycles of PCR, theaqueous solution droplets containing the template DNAs and anotheraqueous solution droplets containing no template DNAs are united bycentrifugation so that secondary PCR amplification is performed byprimers present in the aqueous solution droplets containing no templateDNAs. However, this technique has several difficulties in actualapplications. For example, use of the magnetic stirrer bar wheneverpreparing a water-in-oil emulsion for infectious disease diagnosis isinconvenient and increases contamination occurrence. Furthermore, theaqueous solution droplets have different sizes, which may cause areproducibility problem. Still furthermore, in a case where theconcentration of the template DNAs is very low, several repeatedexperiments are required. In addition, there are disadvantages in that aPCR solution has a large volume of about 50 μl and a quantitative PCR isimpossible.

Meanwhile, a traditional PCR shows the qualitative results of amplifiedDNAs by an electrophoresis at the end-point of the PCR reaction, but hasmany problems such as inaccuracy of the quantitative detection of DNAs.In this regard, a Real-Time PCR was developed to allow for thequantitative detection of amplified DNAs by detecting the intensity offluorescent light, which is in proportional to the concentration of theamplified DNAs, using an optical detection system.

However, in the case of performing the quantitative detection ofamplified DNAs using a typical Real-Time PCR, there are required threeor more repeated experiments using a negative control, a positivecontrol, and at least three standard samples with differentconcentrations, which requires the use of a large number of reactors. Itis impossible to provide such a large number of reactors for a micro PCRchip. To have such a large number of reactors, a larger-sized chip isrequired.

SUMMARY OF THE INVENTION

The present invention provides a method for amplifying nucleic acids inwhich a fluid and an aqueous solution containing PCR reactants areinjected into a reaction vessel via different inlet channels to formaqueous solution droplets surrounded with the fluid in the reactionvessel, the fluid and the aqueous solution being phase-separated fromeach other, and amplification of the nucleic acids in the aqueoussolution droplets is performed.

The present invention also provides an apparatus for amplifying nucleicacids in which the method for amplifying the nucleic acids is performed.

According to an aspect of the present invention, there is provided amethod for amplifying nucleic acids, which includes: introducing into areaction vessel via different inlet channels a reactant aqueous solutioncontaining reactants for nucleic acid amplification and a fluid that isphase-separated from the reactant aqueous solution and does notparticipate in amplification reaction, creating a plurality of reactantaqueous solution droplets surrounded by the fluid by contacting thereactant aqueous solution with the fluid in the reaction vessel, andamplifying the nucleic acids in the reactant aqueous solution droplets.

The fluid may be at least one selected from silicon oil, mineral oil,perfluorinated oil, hydrocarbon oil, and vegetable oil.

The operation of amplifying the nucleic acids may be performed bypolymerase chain reaction (PCR). The operation of amplifying the nucleicacids may also be performed by ligase chain reaction (LCR),strand-displacement amplification (SDA), nucleic acid sequence-basedamplification (NASBA), transcription-mediated amplification (TMA), orloop-mediated isothermal amplification (LAMP).

The operation of amplifying the nucleic acids may be performed by PCR inwhich the entire surface of a substrate is repeatedly heated and cooledto a predetermined temperature.

The operation of amplifying the nucleic acids may be performed bycontinuous-flow PCR in which a substrate is heated to have at least twodifferent temperature areas and the reactant aqueous solution dropletsalternately and repeatedly pass through the different temperature areas.In this case, the substrate may be heated to have at least two differenttemperature areas using a plurality of independently controllableheaters.

For real-time PCR, the operation of introducing the reactant aqueoussolution and the fluid into the reaction vessel may further includeintroducing into the reaction vessel at least one aqueous solution of anegative control and a positive control for quantitative assay and astandard sample aqueous solution via respective inlet channels differentfrom the inlet channels for the introduction of the reactant aqueoussolution and the fluid, and the operation of creating the reactantaqueous solution droplets may further include creating a plurality ofcontrol aqueous solution droplets and a plurality of standard sampleaqueous solution droplets by contacting the at least one aqueoussolution of the negative control and the positive control and thestandard sample aqueous solution with the fluid in the reaction vessel.

For multiplex PCR, in the operation of introducing the reactant aqueoussolution and the fluid into the reaction vessel, at least two reactantaqueous solutions containing at least two primers may be introduced intothe reaction vessel via respective inlet channels.

For hot start PCR, in the operation of introducing the reactant aqueoussolution and the fluid into the reaction vessel, a nucleic acid aqueoussolution, a common component containing polymerase and dNTP, and primerset may be introduced into respective inlet channels and mixed beforebeing introduced into the reaction vessel to form the reactant aqueoussolution.

For single molecule PCR, the reaction vessel may include two primaryreaction channels and a single secondary reaction channel connected to aterminal end of each of the two primary reaction channels, and theoperation of introducing the reactant aqueous solution and the fluidinto the reaction vessel, the operation of creating the reactant aqueoussolution droplets, and the operation of amplifying the nucleic acids maybe performed in each of the two primary reaction channels. In this case,after the operation of amplifying the nucleic acids, the method mayfurther include combining the reactant aqueous solution droplets in astarting end of the secondary reaction channel to create a plurality ofcombined reactant aqueous solution droplets in the secondary reactionchannel and amplifying the nucleic acids in the combined reactantaqueous solution droplets.

The reaction vessel may include at least one reaction chamber. In thiscase, after the operation of amplifying the nucleic acids in thereactant aqueous solution droplets, the method may further includecombining the reactant aqueous solution droplets in the reaction chamberand amplifying the nucleic acids in the combined reactant aqueoussolution droplets. The operation of combining the reactant aqueoussolution droplets may be carried out by centrifugation.

According to another aspect of the present invention, there is providedan apparatus for amplifying nucleic acids, which includes: a substrate;a reaction vessel formed inside of the substrate; at least one firstinlet channel formed inside the substrate, connected to an end of thereaction vessel, and allowing introduction of a reactant aqueoussolution containing reactants for nucleic acid amplification into thereaction vessel; a second inlet channel formed inside the substrate,connected to the end of the reaction vessel, and allowing introductionof a fluid that is phase-separated from the reactant aqueous solutionand does not participate in amplification reaction into the reactionvessel; and a heating unit installed on the substrate in such a way tothermally contact with the substrate and heating the substrate, thereactant aqueous solution contacting with the fluid to create aplurality of reactant aqueous solution droplets surrounded by the fluidin the reaction vessel and the nucleic acids being amplified in thereactant aqueous solution droplets.

The substrate may have a hydrophobic surface property. The substrate maybe made of poly(dimethylsiloxane) (PDMS), silicon, silicon dioxide,plastic, or glass. The substrate may include a lower substrate and anupper substrate, and the reaction vessel, the first inlet channel, andthe second inlet channel may be formed between the lower substrate andthe upper substrate.

For continuous-flow PCR, the reaction vessel may be a serpentinereaction channel, the first inlet channel and the second inlet channelmay be connected to a staring end of the reaction channel, and an outletport for releasing the fluid and an amplification product may beconnected to a terminal end of the reaction channel.

The heating unit may be installed at a lower surface of the substrateand include at least two heaters for heating the substrate to allow thesubstrate to have at least two different temperature areas.

The heating unit may include a plurality of independently controllableheaters installed at a lower surface of the substrate to heat thesubstrate so that the substrate has at least two temperature areas.

The reaction channel may alternately and repeatedly pass through thetemperature areas.

The second inlet channel may linearly extend from the starting end ofthe reaction channel and the first inlet channel may be formedperpendicularly to the second inlet channel.

For real-time PCR, the apparatus may further include a third inletchannel formed inside the substrate, connected to the end of thereaction channel, and allowing introduction of at least one selectedfrom a negative control aqueous solution and a positive control aqueoussolution for quantitative assay into the reaction channel and a fourthinlet channel formed inside the substrate, connected to the end of thereaction channel, and allowing introduction of a standard sample aqueoussolution for quantitative assay into the reaction channel.

The fourth inlet channel may be connected to at least two standardsample inlet channels for introduction of standard samples and adistilled water inlet channel for diluting the standard samples to apredetermined concentration. The fourth inlet channel may have aserpentine shape.

For multiplex PCR, the apparatus may include at least two first inletchannels and the first inlet channels may be correspondingly connectedto at least two primer inlet channels for introduction of at least twodifferent primers into the first inlet channels. In this case, each ofthe first inlet channels may have a serpentine shape.

For hot start PCR, the first inlet channel may be connected to a primerinlet channel for introduction of a primer into the first inlet channeland a common component inlet channel for introduction of a commoncomponent including polymerase and dNTP into the first inlet channel. Inthis case, a portion of a starting end side of the reaction channel mayhave a serpentine shape.

For single molecule PCR, the reaction vessel may include two serpentineprimary reaction channels and a single serpentine secondary reactionchannel connected to a terminal end of each of the two primary reactionchannels, and the first inlet channel and the second inlet channel maybe connected to starting ends of the two primary reaction channels. Inthis case, the heating unit may include a plurality of independentlycontrollable heaters installed at a lower surface of the substrate toprovide two temperature areas for each of the two primary reactionchannels and the single secondary reaction channel.

The reaction vessel may be a reaction chamber, the first inlet channeland the second inlet channel may be connected to an end of the reactionchamber, and an outlet channel for releasing the fluid and anamplification product may be connected to the other end of the reactionchamber. In this case, the apparatus may include a plurality of reactionchambers, first inlet channels, second inlet channels, and outletchannels. The heating unit may include a single heater installed at alower surface of the substrate and heating the entire surface of thesubstrate to a predetermined temperature.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features and advantages of the present inventionwill become more apparent by describing in detail exemplary embodimentsthereof with reference to the attached drawings in which:

FIG. 1A is a schematic plan view of a nucleic acid amplificationapparatus according to a first embodiment of the present invention;

FIG. 1B is an exploded perspective view of the nucleic acidamplification apparatus of FIG. 1A;

FIG. 2 is a schematic plant view of a nucleic acid amplificationapparatus according to a second embodiment of the present invention;

FIGS. 3 and 4 are respectively a charged coupled device (CCD) image of afluorescent signal emitted from each aqueous solution droplet and anintensity of fluorescent signal versus the number of cycles, when PCR isperformed using the nucleic acid amplification apparatus according tothe second embodiment of the present invention;

FIG. 5 is a schematic plan view of a nucleic acid amplificationapparatus according to a third embodiment of the present invention;

FIG. 6 is a schematic plan view of a nucleic acid amplificationapparatus according to a fourth embodiment of the present invention;

FIG. 7 is a schematic plan view of a nucleic acid amplificationapparatus according to a fifth embodiment of the present invention;

FIG. 8 is a schematic plan view of a nucleic acid amplificationapparatus according to a sixth embodiment of the present invention;

FIGS. 9A and 9B are schematic plan views that illustrate respectively afirst PCR step and a second PCR step, in a nucleic acid amplificationapparatus according to a seventh embodiment of the present invention;

FIGS. 10A and 10B are schematic sectional views of a nucleic acidamplification apparatus for illustrating two-dimensional simulationconditions;

FIG. 11 is a view that illustrates a temperature profile versus timewhen cooling is performed from 95° C. to 60° C. during thetwo-dimensional simulation according to the simulation conditions ofFIG. 10A;

FIG. 12 is a graph that illustrates a change in water temperature versustime in a two-dimensional simulation according to the conditionspresented in Table 2;

FIG. 13 is a graph that illustrates the temperature change values of thegraph of FIG. 12 normalized by a total temperature change value withrespect to time;

FIG. 14 is a graph that illustrates a standard deviation of watertemperature versus time in a two-dimensional simulation according to theconditions presented in Table 2;

FIG. 15 is a graph that illustrates a ratio of the standard deviation ofwater temperature of the graph of FIG. 14 with respect to the totaltemperature change value;

FIG. 16 is a schematic view that illustrates an amplification apparatusfor three-dimensional simulation;

FIG. 17 is a view that illustrates a temperature profile of heater areasof the amplification apparatus versus time when a three-dimensionalsimulation is performed according to the conditions shown in FIG. 16;

FIG. 18 is a graph that illustrates a temperature profile of the insideof a channel along the longitudinal direction of the channel;

FIG. 19 is a graph that illustrates a change in water temperature versustime when a three-dimensional simulation is performed according to thecondition shown in FIG. 16;

FIG. 20 is a graph that illustrates the temperature change values of thegraph of FIG. 19 normalized by a total temperature change value withrespect to time; and

FIG. 21 is a graph that illustrates a ratio of the standard deviation ofwater temperature of the graph of FIG. 19 with respect to the totaltemperature change value.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, exemplary embodiments of a method and an apparatus foramplifying nucleic acids according to the present invention will bedescribed in detail with reference to the accompanying drawings. Thesame reference numerals refer to the same components throughout thedrawings.

First Embodiment Continuous-Flow PCR

FIG. 1A is a schematic plan view of a nucleic acid amplificationapparatus according to a first embodiment of the present invention andFIG. 1B is an exploded perspective view of the nucleic acidamplification apparatus of FIG. 1A.

Referring to FIGS. 1A and 1B, the nucleic acid amplification apparatusaccording to the first embodiment of the present invention includes asubstrate 110, a reaction channel 130 formed inside the substrate 110,at least two inlet channels 141 and 151, and a heating unit 120 forheating the substrate 110 to a predetermined temperature.

The substrate 110 may be made of PDMS (Poly Dimethyl Siloxane), silicon,silicon dioxide, plastic, or glass. Preferably, the substrate 110 has ahydrophobic surface property. When the substrate 110 is made of amaterial having no hydrophobic surface property, it is preferable tomodify the surface of the substrate 110 so that the substrate 110 has ahydrophobic surface property. The substrate 110 may include a lowersubstrate 111 and an upper substrate 112 that are bonded to each other,to form the reaction channel 130 inside the substrate 110. Theabove-described construction of the substrate 110 may also be applied toall embodiments that will be described hereinafter.

The reaction channel 130 is formed inside the substrate 110 and servesas a reaction vessel in which amplification of nucleic acids occurs. Indetail, the reaction channel 130 may have an extended serpentine shapeso that it can alternately pass through two areas of the substrate 110that are maintained at different temperatures, as will be describedlater.

Among the two inlet channels 141 and 151, a first inlet channel 141receives an aqueous solution containing reactants for nucleic acidamplification (hereinafter, also simply referred to as “reactant aqueoussolution”) and a second inlet channel 151 receives a fluid that isphase-separated from the aqueous solution and does not participate inamplification.

Each of the two inlet channels 141 and 151 is connected to a startingend of the reaction channel 130. In detail, the second inlet channel 151may linearly extend from the starting end of the reaction channel 130.The first inlet channel 141 may be connected to a joint portion betweenthe reaction channel 130 and the second inlet channel 151 so that it canbe perpendicular to the second inlet channel 151. That is, the reactionchannel 130 and the first and second inlet channels 141 and 151 may beconnected in a “T”-shaped form.

The reaction channel 130 and the first and second inlet channels 141 and151 are formed between the lower substrate 111 and the upper substrate112. That is, the channels 130, 141, and 151 may be formed to apredetermined depth from the upper surface of the lower substrate 111,as shown in FIG. 1B. Alternatively, the channels 130, 141, and 151 mayalso be formed in the upper substrate 112. At least one of the channels130, 141, and 151 may be formed in the lower substrate 111 and the otherchannels may be formed in the upper channel 112. Preferably, thechannels 130, 141, and 151 have a square sectional shape due to easinessof formation, as shown in FIG. 1B. In addition, the channels 130, 141,and 151 may have a polygonal or circular sectional shape.

The first and second inlet channels 141 and 151 are respectivelyconnected to inlet ports 142 and 152 for allowing introduction of thereactant aqueous solution and the fluid from the outside. A terminal endof the reaction channel 130 is connected to an outlet port 162 forreleasing amplification products produced by amplification and thefluid. The inlet ports 141 and 142 and the outlet port 162 may be formedin such a way to vertically pass through the upper substrate 112, asshown in FIG. 1B, but are not limited thereto.

The heating unit 120 is installed on a lower surface of the substrate110 to heat the substrate 110 to a predetermined temperature. Theheating unit 120 may be comprised of two heaters 121, and 122 controlledto different temperatures. The heaters 121 and 122 thermally contactwith the lower surface of the lower substrate 111 and serve to heat thesubstrate 110 to two different temperatures. Therefore, the substrate110 has two areas having different temperatures, for example, a 65° C.area and a 95° C. area. Meanwhile, the heating unit 120 may also becomposed of three or more heaters having different temperatures.

A method for amplifying nucleic acids using the above-described nucleicacid amplification apparatus according to the first embodiment of thepresent invention will now be described.

First, as described above, an aqueous solution containing reactants fornucleic acid amplification and a fluid that is phase-separated from theaqueous solution and does not participate in amplification are insertedinto the reaction channel 130 via the first inlet channel 141 and thesecond inlet channel 151, respectively. At this time, the reactants mayinclude target DNAs, primers, dNTPs, polymerase, and buffers. Theaqueous solution is obtained by dissolving these reactants in distilledwater. The fluid may be silicon oil, mineral oil, perfluorinated oil,hydrocarbon oil, or vegetable oil.

As described above, when the reactant aqueous solution and the fluid,for example an oil 131, are inserted into the reaction channel 130 viathe respective inlet channels 141 and 151 at a constant flow rate, thereactant aqueous solution is surrounded by the oil 131 to form aqueoussolution droplets 132. At this time, since the substrate 110 has ahydrophobic surface property as described above, the reactant aqueoussolution is surrounded by the oil 131 without being adsorbed to an innersurface of the reaction channel 130, to thereby create the aqueoussolution droplets 132.

When a flow rate of the reactant aqueous solution passing through thefirst inlet channel 141 is controlled, the aqueous solution droplets 132can be periodically made up to a constant volume in the reaction channel130.

Here, the aqueous solution droplets 132 surrounded by the oil 131 may becreated using a method suggested by Lamagilov et al. [Rustem F.Lamagilov et al., Angew. Chem. Int. Ed. 2003, 42, No. 7, 768-771].

Each of the aqueous solution droplets 132 created as described aboveserves as a reactor of nucleic acid amplification. That is, nucleic acidamplification occurs in the aqueous solution droplets 132. In detail,the aqueous solution droplets 132 alternately pass through the twotemperature areas of the substrate 110 heated by the heaters 121 and 122during flowing in the reaction channel 130. During this procedure, thereactants for nucleic acid amplification within the aqueous solutiondroplets 132 undergo repeated heating and cooling. Therefore, repeatedcycles of three steps for PCR, i.e., denaturation, annealing, andextension, are performed in the aqueous solution droplets 132. As aresult of the above-described continuous-flow PCR, the concentration ofamplification products in the aqueous solution droplets 132 graduallyincreases.

As described above, according to the present invention, since thereactant aqueous solution passes through the first inlet channel 141with a constant sectional area at a constant flow rate, the aqueoussolution droplets 132 created in the reaction channel 130 have a uniformsize. Furthermore, several tens to several hundreds of the aqueoussolution droplets 132 having a nano-liter (nl) unit volume are easilycreated, which enables repeated experiments in a relatively small-sizedapparatus. Therefore, PCR of about 40 cycles can be performed within avery short time, for example about 10 minutes. In addition, since theaqueous solution droplets 132 are surrounded by the oil 131, acontamination occurrence by adsorption of the reactants for nucleic acidamplification onto the inner surface of the reaction channel 130 can beprevented. At the same time, even when the volume of the reactants isvery small, there is no risk of evaporation of the reactants.

Meanwhile, the first embodiment of the present invention has beenillustrated that the nucleic acid amplification apparatus includes thetwo heaters 121 and 122 for heating the substrate 110 to two differenttemperatures to perform continuous-flow PCR. However, the nucleic acidamplification apparatus according to the first embodiment of the presentinvention may include only one heater. In this case, a PCR technique inwhich the entire surface of the substrate 110 is heated or cooled to thesame temperature may be performed. Alternatively, typical isothermalamplification techniques such as LCR, SDA, NASBA, TMA, and LAMP, insteadof PCR, may be performed. That is, the present invention is not limitedto a PCR technique, but can be applied to various nucleic acidamplification techniques.

Second Embodiment Real-Time PCR

FIG. 2 is a schematic plant view of a nucleic acid amplificationapparatus according to a second embodiment of the present invention.FIGS. 3 and 4 are respectively a charged coupled device (CCD) image of afluorescent signal emitted from each aqueous solution droplet and anintensity of fluorescent signal versus the number of cycles, when PCR isperformed using the nucleic acid amplification apparatus according tothe second embodiment of the present invention.

First, referring to FIG. 2, the nucleic acid amplification apparatusaccording to the second embodiment of the present invention includes asubstrate 110, a reaction channel formed inside the substrate 110, aplurality of inlet channels 241, 243, 245 a, 245 b, 247, 249, and 151,and a heating unit 120 for heating the substrate 110 to a predeterminedtemperature.

Here, the constructions of the substrate 110, the reaction channel 130,and the heating unit 120 are the same as in the first embodiment, andthus, detailed descriptions thereof will be omitted.

The nucleic acid amplification apparatus according to the secondembodiment of the present invention has a construction suitable forreal-time PCR. For this, the inlet channels 241, 243, 245 a, 245 b, 247,249, and 151 are formed inside the substrate 110. In detail, a firstinlet channel 241 for introduction of an aqueous solution containingreactants for nucleic acid amplification and a second inlet channel 151for introduction of the fluid, for example an oil, are connected to astarting end of the reaction channel 130. In addition, a third inletchannel 243 for introduction of a negative control (NTC) aqueoussolution and a fourth inlet channel 249 for introduction of a standardsample aqueous solution are also connected to the starting end of thereaction channel 130. Specifically, the second inlet channel 151 maylinearly extend from the starting end of the reaction channel 130. Thefirst, third, and fourth inlet channels 241, 243, and 249 may beseparated from each other by a predetermined distance and connectedperpendicularly to the reaction channel 130. The first inlet channel 241and the third inlet channel 243 may be respectively connected to inletports 242 and 244 that communicate with the outside.

Meanwhile, a positive control, instead of the negative control, may beintroduced via the third inlet channel 243. An additional inlet channelfor introduction of the positive control may also be formed.

The fourth inlet channel 249 may be connected to two standard sampleinlet channels 245 a and 245 b for introduction of two standard samplesand a distilled water inlet channel 247 for introduction of distilledwater to dilute the standard samples to a predetermined concentration.The two standard sample inlet channels 245 a and 245 b and the distilledwater inlet channel 247 are respectively connected to inlet ports 246 a,246 b, and 248 that communicate with the outside. According to thisconstruction, the standard samples are diluted with the distilled waterin the fourth inlet channel 249 to form a standard sample aqueoussolution with a predetermined concentration. In this regard, adjustmentof the flow rates of the standard samples and the distilled waterenables easy formation of the standard sample aqueous solution ofdifferent concentrations. At this time, it is preferable to form thefourth inlet channel 249 with a serpentine shape so as to promotesufficient mixing of the standard samples and the distilled water.

In the above-described nucleic acid amplification apparatus according tothe second embodiment of the present invention, when the reactantaqueous solution, the NTC aqueous solution, and the standard sampleaqueous solution are respectively introduced via the first, third, andfourth inlet channels 241, 243, and 249 while an oil 131 is introducedvia the second inlet channel 151, reactant aqueous solution droplets 232surrounded by the oil 131, NTC aqueous solution droplets 233, andstandard sample aqueous solution droplets 234 are created in thereaction channel 130, as described in the first embodiment. At thistime, when the aqueous solutions are alternately introduced at apredetermined time interval, the reactant aqueous solution droplets 232,the NTC aqueous solution droplets 233, and the standard sample aqueoussolution droplets 234 are alternately created in the reaction channel130, as shown in FIG. 2. In this way, when the aqueous solution droplets232, 233, and 234 thus created alternately pass through two temperatureareas of the substrate 110 heated by the two heaters 121 and 122 duringflowing in the reaction channel 130, continuous-flow PCR is performed inthe aqueous solution droplets 232, 233, and 234.

When a fluorescent dye is contained in each of the aqueous solutions,quantitative analysis of nucleic acids is possible by detecting afluorescent signal emitted from each of the aqueous solution droplets232, 233, and 234 using an optical detection system. That is, wheninitially created droplets among the reactant aqueous solution droplets232 reach a terminal end of the reaction channel 130, a CCD image of afluorescent signal using a CCD camera as shown in FIG. 3 can beobtained. Referring to FIG. 3, a fluorescent signal is emitted from eachof the aqueous solution droplets flowing in a predetermined distance inthe reaction channel. Therefore, a more simple and accurate quantitativeanalysis of fluorescent signals is possible, as compared to aconventional PCR technique in which a fluorescent signal is emitted fromthe entire surface of a reaction channel. Based on the CCD image thusobtained, a real-time PCR curve as shown in FIG. 4 can be obtained.

As described above, according to the second embodiment of the presentinvention, a real-time PCR curve can be obtained by one-pot imageanalysis in a single PCR apparatus, which enables quantitative analysisof nucleic acids. Furthermore, since the reactant aqueous solution, theNTC aqueous solution, and the standard sample aqueous solution areintroduced in the single reaction channel 130 and then experiments areperformed at the same time, a smaller PCR apparatus can be used and atime required for real-time PCR can be significantly reduced. Inaddition, since adjustment of the flow rates of the standard samples andthe distilled water enables easy formation of the standard sampleaqueous solution of different concentrations, more accurate quantitativeanalysis is possible.

Third Embodiment Multiplex PCR

FIG. 5 is a schematic plan view of a nucleic acid amplificationapparatus according to a third embodiment of the present invention.

Referring to FIG. 5, in the nucleic acid amplification apparatusaccording to the third embodiment of the present invention, theconstructions of a substrate 110, a reaction channel 130, and a heatingunit 120 are the same as in the first embodiment, and thus, detaileddescriptions thereof will be omitted.

The nucleic acid amplification apparatus according to the thirdembodiment of the present invention has a construction suitable formultiplex PCR for simultaneous analysis of multiple genes in a singleapparatus. For this, the substrate 110 is formed with a plurality of,for example, three first inlet channels 341 for introduction of reactantaqueous solutions, a plurality of primer inlet channels 343 forintroduction of three different primers A, B, and C into the first inletchannels 341, and a single second inlet channel 151 for oilintroduction. The second inlet channel 151 may linearly extend from astarting end of the reaction channel 130. The first inlet channels 341may be separated from each other by a predetermined distance andconnected perpendicularly to the reaction channel 130. The primer inletchannels 343 are correspondingly connected to the first inlet channels341. The first inlet channels 341 and the primer inlet channels 343 maybe respectively connected to inlet ports 342 and 344 that communicatewith the outside.

In the above-described nucleic acid amplification apparatus according tothe third embodiment of the present invention, when the three differentprimers A, B, and C are introduced into the first inlet channels 341 viathe three primer inlet channels 343, respectively, the reactant aqueoussolutions in the first inlet channels 341 contain different primers.Preferably, the first inlet channels 341 are formed in a serpentineshape so that the reactant aqueous solutions and the primers aresufficiently mixed.

Next, when the reactant aqueous solutions are introduced into thereaction channel 130 via the first inlet channels 341 while an oil 131is introduced into the reaction channel 130 via the second inlet channel151, aqueous solution droplets 332 a, 332 b, and 332 c surrounded by theoil 131 and containing the different primers A, B, and C are created inthe reaction channel 130. At this time, when the reactant aqueoussolutions containing the different primers A, B, and C are alternatelyintroduced via the first inlet channels 341 at a predetermined timeinterval, the aqueous solution droplets 332 a, 332 b, and 332 c may bealternately created in the reaction channel 130, as shown in FIG. 5.

As described above, according to the third embodiment of the presentinvention, it is possible to perform multiplex PCR in which multiplegenes are simultaneously analyzed in a single micro-sized apparatus.Furthermore, since the aqueous solution droplets 332 a, 332 b, and 332 ccontaining different primers are surrounded by the oil 131, crosscontamination of the reactant aqueous solutions can be prevented.

Fourth Embodiment Hot Start PCR

FIG. 6 is a schematic plan view of a nucleic acid amplificationapparatus according to a fourth embodiment of the present invention.

Referring to FIG. 6, in the nucleic acid amplification apparatusaccording to the fourth embodiment of the present invention, theconstructions of a substrate 110, a reaction channel 130, and a heatingunit 120 are the same as in the first embodiment, and thus, detaileddescriptions thereof will be omitted.

The nucleic acid amplification apparatus according to the fourthembodiment of the present invention has a construction suitable for hotstart PCR. For this, the substrate 110 is formed with a first inletchannel 441 for introduction of a reactant aqueous solution, a primerinlet channel 443 for introduction of primers into the first inletchannel 441, a common component inlet channel for introduction of commonreaction components, for example, polymerase, dNTPs, and buffer, intothe first inlet channel 441, and a second inlet channel 151 for oilintroduction. The second inlet channel 151 may linearly extend from astarting end of the reaction channel 130. The first inlet channel 441may be connected perpendicularly to the reaction channel 130. The primerinlet channel 443 and the common component inlet channel 445 areconnected to a central portion of the first inlet channel 441. The firstinlet channel 441, the primer inlet channel 443, and the commoncomponent inlet channel 445 may be respectively connected to inlet ports442, 444, and 446 that communicate with the outside.

In the above-described nucleic acid amplification apparatus according tothe fourth embodiment of the present invention, when the primers and thecommon reaction components are respectively introduced into the primerinlet channel 443 and the common component inlet channel 445 while atarget DNA aqueous solution is introduced into the first inlet channel441, the target DNA aqueous solution, the primers, and the commonreaction components are mixed in a central portion of the first inletchannel 441 to form a reactant aqueous solution for nucleic acidamplification. The reactant aqueous solution thus formed is introducedinto the reaction channel 130 via the first inlet channel 441.Therefore, a plurality of reactant aqueous solution droplets 432surrounded by an oil 131 are created in the reaction channel 130. Whenthe reactant aqueous solution droplets 432 alternately pass through twotemperature areas of the substrate 110 heated by two heaters 121 and 122during flowing in the reaction channel 130, continuous-flow PCR isperformed in the reactant aqueous solution droplets 432.

Here, it is preferable to form a portion of a starting end side of thereaction channel 130 with a serpentine shape so that the reactants inthe aqueous solution droplets 432 are sufficiently heated at apredetermined temperature before the aqueous solution droplets 432 reacha melting temperature of a first cycle.

As described above, according to the fourth embodiment of the presentinvention, since the target DNA aqueous solution, the primers, and thecommon components are mixed to form the reactant aqueous solution fornucleic acid amplification immediately before the first cycle of PCR,hot start PCR is possible. Therefore, formation of nonspecific productssuch as primer-dimer that may be generated at a low temperature prior toinitiation of PCR can be prevented.

Fifth Embodiment Temperature Programmable Continuous-Flow PCR

FIG. 7 is a schematic plan view of a nucleic acid amplificationapparatus according to a fifth embodiment of the present invention.

Referring to FIG. 7, in the nucleic acid amplification apparatusaccording to the fifth embodiment of the present invention, theconstructions of a substrate 110, a reaction channel 130, and inletchannels 441, 443, 445, and 151 are the same as in the fourth embodimentof the present invention, and thus, detailed descriptions thereof willbe omitted.

The nucleic acid amplification apparatus according to the fifthembodiment of the present invention includes a heating unit 520 composedof a plurality of independently controllable heaters 5211 through 521 n.In detail, the heaters 5211 through 521 n are disposed in parallel andthermally contact with a lower surface of the substrate 110. The heaters5211 through 521 n serve to heat corresponding portions of the substrate110 to a predetermined temperature.

Based on the above-described construction, the temperature condition ofeach of the heaters 5211 through 521 n can be changed as necessary,which allows the substrate 110 to have differentially programmedtemperature areas. For example, when the heaters 5211 through 521 n aredivided into three groups that are adjusted to different temperatures,the substrate 110 can have three different temperature areas T1, T2, andT3. Therefore, this embodiment has an advantage in which a nucleic acidamplification experiment can be performed in a single apparatus underdifferent temperature conditions.

The above-described heating unit 520 may also be applied to the nucleicacid amplification apparatuses of the first through fourth embodiments.

Sixth Embodiment Single Molecule PCR

FIG. 8 is a schematic plan view of a nucleic acid amplificationapparatus according to a sixth embodiment of the present invention.

Referring to FIG. 8, the nucleic acid amplification apparatus accordingto the sixth embodiment of the present invention has a constructionsuitable for single molecule PCR. For this, a substrate 110 is formedwith two primary reaction channels 631 and 632 for primary PCR, a singlesecondary reaction channel 633 for secondary PCR, a first inlet channel641 for introduction of a reactant aqueous solution into the two primaryreaction channels 631 and 632, and two second inlet channels 651 and 653for introduction of an oil into the primary reaction channels 631 and632, respectively.

The two second inlet channels 651 and 653 are respectively connected tostarting ends of the two primary reaction channels 631 and 632. Thefirst inlet channel 641 is commonly connected to the starting ends ofthe two primary reaction channels 631 and 632. The first inlet channel641 and the two second inlet channels 651 and 653 may be respectivelyconnected to inlet ports 642, 652, and 654 that communicate with theoutside.

Meanwhile, a single second inlet channel may also be commonly connectedto the two primary reaction channels 631 and 632. Two first inletchannels may also be correspondingly connected to the two primaryreaction channels 631 and 632.

Terminal ends of the two primary reaction channels 631 and 632 areconnected to a starting end of the secondary reaction channel 633. Aterminal end of the secondary reaction channel 633 is connected to anoutlet port 634 that communicates with the outside.

A heating unit is disposed on a lower surface of the substrate 110 to beheated. The heating unit thermally contacts with the lower surface ofthe substrate 110, as shown in FIG. 8, and may includes a plurality ofindependently controllable heaters. The heaters thermally contact withcorresponding lower surface portions of the substrate 110 to allow thesubstrate 110 to have a plurality of different temperature areas. Forexample, as shown in FIG. 8, the heating unit may be composed of fiveheaters 621 through 625 so that each of the primary reaction channels631 and 632 and the secondary reaction channel 633 has two differenttemperature areas.

In the above-described nucleic acid amplification apparatus according tothe sixth embodiment of the present invention, aqueous solution droplets662 a and 662 b surrounded by an oil 661 are created in the two primaryreaction channels 631 and 632. At this time, when the concentration ofnucleic acids contained in the reactant aqueous solution is very low,first aqueous solution droplets 662 a containing a trace or noconcentration of nucleic acids and second aqueous solution droplets 662b containing a relatively high concentration of nucleic acids may becreated in the primary reaction channels 631 and 632. While the firstand second aqueous solution droplets 662 a and 662 b flow in the primaryreaction channels 631 and 632, primary PCR is performed in the first andsecond aqueous solution droplets 662 a and 662 b. During this procedure,most primers in the second aqueous solution droplets 662 b containing arelatively high concentration of nucleic acids are consumed. On theother hand, most primers in the first aqueous solution droplets 662 aremain unreacted.

The first and second aqueous solution droplets 662 a and 662 b arecombined in the secondary reaction channel 633 after the primary PCR iscompleted. At this time, the first aqueous solution droplets 662 a andthe second aqueous solution droplets 662 b may be combined amongthemselves or may form third aqueous solution droplets 662 c. In thecase of the latter, since nucleic acids of the second aqueous solutiondroplets 662 b meet with primers of the first aqueous solution droplets662 a, the nucleic acids and the primers coexist in the third aqueoussolution droplets 662 c. Therefore, while the third aqueous solutiondroplets 662 c flow in the secondary reaction channel 633, secondary PCRis performed.

As described above, according to this embodiment of the presentinvention, two-step PCR is performed in a single apparatus. Therefore,sensitivity of PCR can be remarkably enhanced. Furthermore, sinceseveral tens to several hundreds of the aqueous solution droplets 662 aand 662 b can be continuously created in a single apparatus, traceamounts of nucleic acids can be sufficiently amplified even by onlyone-pot experiment. In addition, since the aqueous solution droplets 662a, 662 b, and 662 c used as reactors have a very small volume,sensitivity in amplification of trace amounts of nucleic acids issignificantly enhanced.

Meanwhile, the above-described single molecule PCR technique usingtwo-step PCR may also be applied to a nucleic acid amplificationapparatus of a chamber shape as will be described later, in addition tothe nucleic acid amplification apparatus of a channel shape.

Seventh Embodiment Single Molecule PCR

FIGS. 9A and 9B are schematic plan views that illustrate respectively afirst PCR step and a second PCR step, in a nucleic acid amplificationapparatus according to a seventh embodiment of the present invention.

First, referring to FIG. 9A, the nucleic acid amplification apparatusaccording to the seventh embodiment of the present invention includes asubstrate 110, a reaction chamber 730 formed inside the substrate 110,inlet channels 741 and 751, an outlet channel 761, and a heating unitfor heating the substrate 110 to a predetermined temperature.

The construction of the substrate 110 is the same as in the firstembodiment.

The reaction chamber 730 is formed inside the substrate 110 and servesas a reaction vessel in which nucleic acid amplification occurs. Thereaction chamber 730 may be polygonal or circular and may be formed inseveral numbers in the single substrate 110.

An end of the reaction chamber 730 is connected to a first inlet channel741 for introduction of a reactant aqueous solution and a second inletchannel 751 for introduction of a fluid, for example an oil 731. Theother end of the reaction chamber 730 is connected to the outlet channel761 for releasing amplification products and the oil 731 from thereaction chamber 730. The first and second inlet channels 741 and 751are respectively connected to inlet ports 742 and 752 that communicatewith the outside. The outlet channel 761 is connected to an outlet port762 that communicates with the outside.

The heating unit is disposed on a lower surface of the substrate 110 andincludes a heater 721 for heating the substrate 110 to a predeterminedtemperature.

A method for amplifying nucleic acids using the above-described nucleicacid amplification apparatus according to the seventh embodiment of thepresent invention will now be described.

First, as described above, the reactant aqueous solution and the fluid,for example, the oil 731 are introduced into the reaction chamber 730via the first inlet channel 741 and the second inlet channel 751,respectively, to create a plurality of aqueous solution droplets 732 aand 732 b surrounded by the oil 731 in the reaction chamber 730. At thistime, adjustment of the flow rate of the reactant aqueous solutionpassing through the first inlet channel 741 allows the aqueous solutiondroplets 732 a and 732 b created in the reaction chamber 730 to have auniform volume. During this procedure, when the concentration of nucleicacids contained in the reactant aqueous solution is very low, firstaqueous solution droplets 732 a containing a trace or no concentrationof nucleic acids and second aqueous solution droplets 732 b containing arelatively high concentration of nucleic acids can be created in thereaction chamber 730.

Next, when the substrate 110 is periodically heated and cooled by theheater 721, a first PCR step is performed in the aqueous solutiondroplets 732 a and 732 b. During the procedure, most primers of thesecond aqueous solution droplets 732 b containing a relatively highconcentration of nucleic acids are consumed. On the other hand, mostprimers of the first aqueous solution droplets 732 a remain unreacted.

Referring to FIG. 9B, after the first PCR step is completed, the firstaqueous solution droplets 732 a and the second aqueous solution droplets732 b are combined by centrifugation to create third aqueous solutiondroplets 732 c with a larger volume. As a result, nucleic acids of thesecond aqueous solution droplets 732 b meet with primers of the firstaqueous solution droplets 732 a. Therefore, nucleic acids and primerscoexist in the third aqueous solution droplets 732 c.

In this state, when the substrate 110 is periodically heated and cooledby the heater 721, a second PCR step is performed in the third aqueoussolution droplets 732 c.

The nucleic acid amplification apparatus according to the seventhembodiment of the present invention has been illustrated in terms of aPCR technique. However, the nucleic acid amplification apparatusaccording to the seventh embodiment of the present invention may also beapplied to typical isothermal amplification techniques such as LCR, SDA,NASBA, TMA, and LAMP.

EXPERIMENTAL EXAMPLES

With respect to the continuous-flow PCR according to the first throughsixth embodiments of the present invention, since a reactant aqueoussolution and an oil, which are different in thermal conductivity, flowin a reaction channel, it is necessary to preset a flow rate of thereactant aqueous solution and the oil. In addition, design parameterssuch as the thickness of a substrate and a gap between a heater and thereaction channel have to be preset.

Hereinafter, there will be provided parameters required for designing anucleic acid amplification apparatus, which are determined bysimulations based on thermal characteristics of a substrate, and areactant aqueous solution and an oil flowing in a reaction channel.

Thermal characteristics of several materials are summarized in Table 1below. TABLE 1 Thermal Specific Thermal Kinematic conductivity Densityheat diffusivity viscosity Material (W/m · K) (kg/m³) (J/kg · k) (m²/s)Rank (m²/s) Si 157 2329 700 9.630E−05 1 Glass 1.13 2520 753 5.955E−07 5Polycarbonate 0.2 1200 1250 1.333E−07 7 Thermally Conductive 20 1700 9001.307E−05 3 LCP Water 0.613 997 4179 1.471E−07 6 1.000E−06 SiO₂ 10.42650 745 5.268E−06 4 Pt 71.6 21500 133 2.504E−05 2 Perfluorodecalin0.0677 1930 970 3.613E−08 9 2.94-E−06 (C₁₀F₁₈) PDMS 0.17 1050 13151.231E−07 8LCP: Liquid crystal polymer,PDMS: Poly(dimethylsiloxane)

As shown in Table 1, the thermal characteristics of PDMS andperfluorodecalin (C₁₀F₁₈) are the worst.

In this regard, the following simulations were performed under the worstconditions using a substrate made of PDMS and perfluorodecalin as anoil.

Experimental Example 1 Two-Dimensional Simulation

FIGS. 10A and 10B are schematic sectional views of a nucleic acidamplification apparatus for illustrating two-dimensional simulationconditions.

First, referring to FIG. 10A, a substrate was made of PDMS. A heater wasdisposed on a lower surface of the substrate. The substrate was formedwith a reaction channel in which each of a width Wc and a height Hc was100 μm. A thickness Gp of the substrate between the reaction channel andthe heater was set to 10 μm and a height H_(W) of water in the reactionchannel was set to 90 μm.

A first two-dimensional simulation was performed under theabove-described conditions.

Then, a second two-dimensional simulation was performed with varying theaspect ratio (W_(C)/H_(C)) of the reaction channel and a ratio of H_(W)to H_(C).

Next, referring to FIG. 10B, a third two-dimensional simulation wasperformed in the same manner as shown in FIG. 10A except that thethickness G_(P) of the substrate between the reaction channel and theheater was set to 100 μm.

Meanwhile, the sectional shape of water in the reaction channel willsubstantially approximate to a circular shape due to the surface tensionof water. However, to more easily perform the simulations, it wassupposed that water in the reaction channel of FIGS. 10A and 10B had asquare sectional shape.

Illustrative conditions of the two-dimensional simulations aresummarized in Table 2 below. TABLE 2 Thickness Aspect Ratio of ofsubstrate ratio water height between of to channel Sec- channel andchannel height Temperature Condition tion heater (G_(P)) (W_(C)/H_(C))(H_(W)/H_(C)) Cooling Heating {circle over (1)}  10 μm 1(100/100) 0.595° C. => 30° C. => 60° C. 95° C. {circle over (2)} 0.8 {circle over(3)} 0.9 {circle over (4)} 2(140/70)  0.8 {circle over (5)} 0.9 {circleover (6)} 4(200/50)  0.8 {circle over (7)} 0.9 {circle over (8)} 100 μm1(100/100) 0.9

FIGS. 11 through 15 show results of the two-dimensional simulations.

FIG. 11 illustrates a temperature profile versus time when cooling isperformed from 95° C. to 60° C. during the two-dimensional simulationaccording to the simulation conditions of FIG. 10A.

As shown in FIG. 11, all areas of the nucleic acid amplificationapparatus, i.e., the substrate, and the oil and the water contained inthe reaction channel reached a target temperature within 0.5 seconds.

FIG. 12 is a graph that illustrates a change in water temperature versustime in the two-dimensional simulations according to the conditionspresented in Table 2. Each temperature curve of FIG. 12 represents anaverage value of temperatures of six spots of a water-containing sectionof the reaction channel with respect to time.

As shown in the graph of FIG. 12, when the thickness G_(P) of thesubstrate between the reaction channel and the heater was 10 μm, watertemperature reached a target value within 0.5 seconds by cooling andheating regardless of the aspect ratio of the reaction channel.

Meanwhile, as shown in FIG. 12, a temperature change value betweenheating and cooling is different. In this regard, a graph of FIG. 13shows a temperature change value normalized by total temperature changevalue with respect to time.

It can be seen from the graph of FIG. 13 that a change in temperatureversus time during heating is similar to that during cooling.

The above simulation results show that a method and an apparatus foramplifying nucleic acids according to the present invention provide aremarkably rapid response speed with respect to temperature change,which ensures rapid PCR.

According to the present invention, in temperature change of waterdroplets surrounded by oil, temperature uniformity acts as an importantfactor. The above-described simulations demonstrated that an apparatusand a method for amplifying nucleic acids of the present inventionprovide temperature uniformity.

FIG. 14 is a graph that illustrates a standard deviation of watertemperature versus time in the two-dimensional simulations according tothe conditions presented in Table 2. Each standard deviation curve ofFIG. 14 represents an average value of standard deviations oftemperatures of six spots of a water-containing section of the reactionchannel with respect to time.

As shown in the graph of FIG. 14, when the thickness (G_(P)) of thesubstrate between the reaction channel and the heater was 10 μm, a timefor which standard deviation reached 1° C. or less was 0.3 seconds orless regardless of the aspect ratio of the reaction channel. When thethickness (G_(P)) of the substrate between the reaction channel and theheater was 100 μm, standard deviation reached 1° C. or less within 0.4seconds.

A graph of FIG. 15 shows a ratio of the standard deviation of FIG. 14 toa total temperature change value.

As shown in the graph of FIG. 15, a time required for accomplishingtemperature uniformity of 2% or less of total temperature change valuewas 0.5 seconds or less. This means that when a temperature change of50° C. occurs, a period of time sufficient to accomplish temperatureuniformity of 1° C. or less is 0.5 seconds.

Experimental Example 2 Three-Dimensional Simulation

This Experimental Example was performed to evaluate thermalcharacteristics of a three-dimensional structure that approximates to anactual structure, based on the above-described two-dimensionalsimulations.

FIG. 16 is a schematic view that illustrates an amplification apparatusfor three-dimensional simulation.

Referring to FIG. 16, two heaters are disposed on a lower surface of asubstrate made of PDMS. The substrate is formed with a reaction channel.A water droplet surrounded by oil is present in the reaction channel.

Under this construction, illustrative conditions for three-dimensionalsimulation was the same as that as shown in FIG. 10A. That is, each ofwidth W_(C) and height H_(C) of the reaction channel was set to 100 μmand a thickness G_(P) of the substrate between the reaction channel andthe heaters was set to 10 μm. Each of the height, width, and the lengthof the water droplet was set to 90 μm. A length of a first heater was3,000 μm and a length of a second heater was 1,000 μm.

An initial condition of the three-dimensional simulation was as follows.A temperature of the first heater shown in a right side of FIG. 16 was60° C. and an initial temperature of oil and water droplet in a firstheater area was 95° C. A temperature of the second heater shown in aleft side of FIG. 16 was 95° C. and an initial temperature of oil andwater droplet in a second heater area was 60° C. In this regard, thefirst heater area is an area in which oil and water are cooled from 95°C. to 60° C. and the second heater area is an area in which oil andwater are heated from 60° C. to 95° C.

FIGS. 17 through 21 show results of the three-dimensional simulation.

FIG. 17 illustrates a temperature profile of each area of a nucleic acidamplification apparatus with respect to time when three-dimensionalsimulation is performed according to the conditions shown in FIG. 16.

As shown in FIG. 17, all areas of the nucleic acid amplificationapparatus reached a target temperature within 0.5 seconds.

FIG. 19 is a graph that illustrates a change in water temperature versustime in the three-dimensional simulation performed according to thecondition shown in FIG. 16. Each temperature curve of FIG. 19 representsan average value of temperatures of 18 water-containing spots of areaction channel with respect to time. FIG. 20 is a graph thatillustrates the temperature change value of FIG. 19 normalized by atotal temperature change value with respect to time.

The graphs of FIGS. 19 and 20 show both the results of the two- andthree-dimensional simulations to compare the two results. In the graphsof FIGS. 19 and 20, {circle over (11)} represents a heating curve of thethree-dimensional simulation and {circle over (12)} represents a coolingcurve of the three-dimensional simulation.

From the graphs of FIGS. 19 and 20, it can be seen that the results ofthe three-dimensional simulation are similar to those of thetwo-dimensional simulations.

Meanwhile, to rapidly perform PCR, oil and water droplet must rapidlyreach the temperatures of heaters disposed on a lower surface of areaction channel. In this regard, when two areas with differenttemperatures are adjacent to each other, a spatial temperature profileof the inside of a reaction channel was evaluated.

FIG. 18 is a graph that illustrates a temperature profile of the insideof a reaction channel along the longitudinal direction of the reactionchannel. In FIG. 18, the length “1 mm” of a left side indicates a 95° C.area and the length “3 mm” of a right side indicates a 60° C. area.

As shown in FIG. 18, a temperature area is influenced by anothertemperature area within 0.5 mm distance. This means that when a distancebetween two areas with different temperatures is about 1 mm, the twoareas completely get out of the interaction.

Like the above-described two-dimensional simulations, to evaluatetemperature uniformity of water droplet surrounded by oil in thethree-dimensional simulation, standard deviations of temperatures at 18spots of the water droplet were calculated.

FIG. 21 is a graph that illustrates a ratio of the standard deviation ofwater temperature to the total temperature change value in thethree-dimensional simulation. The graph of FIG. 21 shows both theresults of the two- and three-dimensional simulations to compare the tworesults. In the graph of FIG. 21, {circle over (11)} represents athree-dimensional simulation curve.

As shown in FIG. 21, a time required for accomplishing temperatureuniformity of 2% or less of total temperature change value was 0.5seconds or less. This means that when a temperature change of 50° C.occurs, a period of time sufficient to accomplish temperature uniformityof 1° C. or less is 0.5 seconds.

Based on the above simulation results, response characteristics withrespect to temperature and temperature uniformity are summarized inTable 3 below. TABLE 3 Thickness of substrate Ratio of water CoolingHeating between channel Aspect ratio of height to 90% NorStdev = 90%NorStdev = Simulation and heater channel channel height Target 2% Target2% section (G_(P)) (W_(C)/H_(C)) (H_(W)/H_(C)) (msec) (msec) (msec)(msec) Three  10 μm 1(100/100) 0.9 287 384 262 703 dimensionalsimulation Two  10 μm 1(100/100) 0.5 320 212 316 238 dimensional 0.8 271284 268 325 simulation 0.9 239 292 236 337 2(140/70)  0.8 186 185 184201 0.9 155 190 154 208 4(200/50)  0.8 133 142 132 152 0.9 113 143 112153 100 μm 1(100/100) 0.9 620 440 618 556

As shown in Table 3, both the response characteristics of temperatureand temperature uniformity that are important factors of thermalcharacteristics reached a desired level within one second. Inparticular, when a thickness of a substrate made of PDMS between aheater and a reaction channel was 10 μm, a uniform distribution of atarget temperature was accomplished within 0.5 seconds.

As apparent from the above description, according to the presentinvention, a reactant aqueous solution passes through an inlet channelwith a constant sectional area at a constant flow rate. Therefore,several tens to several hundreds of aqueous solution droplets surroundedby oil and having fine and uniform sizes can be easily created in areaction channel or a reaction chamber, which ensures rapid repeated PCRexperiments in a relatively small-sized apparatus. Furthermore, sincethe aqueous solution droplets are surrounded by oil, a contaminationproblem that may be caused by adsorption of amplification reactants ontothe inside of the reaction channel can be prevented and there is no riskof evaporation even when the reactants have a very small volume.

According to the present invention, a reactant aqueous solution, a NTCaqueous solution, and a standard sample aqueous solution are alltogether introduced into a single reaction channel and then experimentsare simultaneously carried out. Therefore, there are advantages in thata PCR apparatus of the present invention can be miniaturized, relativeto a conventional PCR apparatus, and a time required for real-time PCRcan be remarkably reduced. Furthermore, since a fluorescent signal isemitted from each of the aqueous solution droplets flowing at apredetermined distance in the reaction channel, a quantitative assay ofthe fluorescent signal can be more simply and accurately performed,relative to a conventional PCR technique in which a fluorescent signalis emitted from the entire surface of a reaction channel. Stillfurthermore, a real-time PCR curve can be obtained by one-pot imageassay in a single apparatus, which enables quantitative assay of nucleicacids.

According to the present invention, the structural change of an inletchannel enables hot-start PCR as well as multiplex PCR for simultaneousassay of multiple genes in a single small-sized apparatus.

A nucleic acid amplification apparatus of the present invention caninclude a heating unit composed of a plurality of independentlycontrollable heaters. Therefore, a substrate can have differentiallyprogrammable temperature areas, which enables nucleic acid amplificationexperiments under different temperature conditions in a singleapparatus.

In addition, according to the present invention, single molecule PCR inwhich two-step PCR is performed in a single apparatus is possible,thereby enhancing sensitivity of PCR.

While the present invention has been particularly shown and describedwith reference to exemplary embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the present invention as defined by the following claims.

1. A method for amplifying nucleic acids, which comprises: introducinginto a reaction vessel via different inlet channels a reactant aqueoussolution containing reactants for nucleic acid amplification and a fluidthat is phase-separated from the reactant aqueous solution and does notparticipate in amplification reaction; creating a plurality of reactantaqueous solution droplets surrounded by the fluid by contacting thereactant aqueous solution with the fluid in the reaction vessel; andamplifying the nucleic acids in the reactant aqueous solution droplets.2. The method of claim 1, wherein the fluid is at least one selectedfrom the group consisting of silicon oil, mineral oil, perfluorinatedoil, hydrocarbon oil, and vegetable oil.
 3. The method of claim 1,wherein the operation of amplifying the nucleic acids is performed byone selected from the group consisting of polymerase chain reaction(PCR), ligase chain reaction (LCR), strand-displacement amplification(SDA), nucleic acid sequence-based amplification (NASBA),transcription-mediated amplification (TMA), and loop-mediated isothermalamplification (LAMP).
 4. The method of claim 3, wherein the operation ofamplifying the nucleic acids is performed by PCR.
 5. The method of claim4, wherein the operation of amplifying the nucleic acids is performed byPCR in which the entire surface of a substrate is repeatedly heated andcooled to a predetermined temperature.
 6. The method of claim 4, whereinthe operation of amplifying the nucleic acids is performed bycontinuous-flow PCR in which a substrate is heated to have at least twodifferent temperature areas and the reactant aqueous solution dropletsalternately and repeatedly pass through the different temperature areas.7. The method of claim 6, wherein the substrate is heated to have atleast two different temperature areas using a plurality of independentlycontrollable heaters.
 8. The method of claim 4, wherein the operation ofintroducing the reactant aqueous solution and the fluid into thereaction vessel further comprises introducing into the reaction vesselat least one aqueous solution of a negative control and a positivecontrol for quantitative assay and a standard sample aqueous solutionvia respective inlet channels different from the inlet channels for theintroduction of the reactant aqueous solution and the fluid, and theoperation of creating the reactant aqueous solution droplets furthercomprises creating a plurality of control aqueous solution droplets anda plurality of standard sample aqueous solution droplets by contactingthe at least one aqueous solution of the negative control and thepositive control and the standard sample aqueous solution with the fluidin the reaction vessel.
 9. The method of claim 8, wherein in theoperation of introducing the reactant aqueous solution and the fluidinto the reaction vessel, the standard sample aqueous solution isadjusted to at least two concentrations and then introduced into thereaction vessel.
 10. The method of claim 4, wherein in the operation ofintroducing the reactant aqueous solution and the fluid into thereaction vessel, at least two reactant aqueous solutions containing atleast two primers are introduced into the reaction vessel via respectiveinlet channels.
 11. The method of claim 4, wherein in the operation ofintroducing the reactant aqueous solution and the fluid into thereaction vessel, a nucleic acid aqueous solution, a common componentcontaining polymerase and dNTP, and a primer are introduced intorespective inlet channels and mixed before being introduced into thereaction vessel to form the reactant aqueous solution.
 12. The method ofclaim 1, wherein the reaction vessel comprises two primary reactionchannels and a single secondary reaction channel connected to a terminalend of each of the two primary reaction channels, and the operation ofintroducing the reactant aqueous solution and the fluid into thereaction vessel, the operation of creating the reactant aqueous solutiondroplets, and the operation of amplifying the nucleic acids areperformed in each of the two primary reaction channels, and whereinafter the operation of amplifying the nucleic acids, the method furthercomprises: combining the reactant aqueous solution droplets in astarting end of the secondary reaction channel to create a plurality ofcombined reactant aqueous solution droplets in the secondary reactionchannel; and amplifying the nucleic acids in the combined reactantaqueous solution droplets.
 13. The method of claim 1, wherein thereaction vessel comprises at least one reaction chamber, and whereinafter the operation of amplifying the nucleic acids in the reactantaqueous solution droplets, the method further comprises: combining thereactant aqueous solution droplets in the reaction chamber; andamplifying the nucleic acids in the combined reactant aqueous solutiondroplets.
 14. The method of claim 13, wherein the operation of combiningthe reactant aqueous solution droplets is carried out by centrifugation.15. An apparatus for amplifying nucleic acids, which comprises: asubstrate; a reaction vessel formed inside of the substrate; at leastone first inlet channel formed inside the substrate, connected to an endof the reaction vessel, and allowing introduction of a reactant aqueoussolution containing reactants for nucleic acid amplification into thereaction vessel; a second inlet channel formed inside the substrate,connected to the end of the reaction vessel, and allowing introductionof a fluid that is phase-separated from the reactant aqueous solutionand does not participate in amplification reaction into the reactionvessel; and a heating unit installed on the substrate and heating thesubstrate, the reactant aqueous solution contacting with the fluid tocreate a plurality of reactant aqueous solution droplets surrounded bythe fluid in the reaction vessel and the nucleic acids being amplifiedin the reactant aqueous solution droplets.
 16. The apparatus of claim15, wherein the fluid is at least one selected from the group consistingof silicon oil, mineral oil, perfluorinated oil, hydrocarbon oil, andvegetable oil.
 17. The apparatus of claim 15, wherein the substrate hasa hydrophobic surface property.
 18. The apparatus of claim 17, whereinthe substrate is made of one material selected from the group consistingof poly(dimethylsiloxane) (PDMS), silicon, silicon dioxide, plastic, andglass.
 19. The apparatus of claim 15, wherein the substrate comprises alower substrate and an upper substrate, and the reaction vessel, thefirst inlet channel, and the second inlet channel are formed between thelower substrate and the upper substrate.
 20. The apparatus of claim 15,wherein the nucleic acids are amplified in the reactant aqueous solutiondroplets by one selected from the group consisting of PCR, LCR, SDA,NASBA, TMA, and LAMP.
 21. The apparatus of claim 20, wherein the nucleicacids are amplified by PCR in the reactant aqueous solution droplets.22. The apparatus of claim 21, wherein the reaction vessel is aserpentine reaction channel, the first inlet channel and the secondinlet channel are connected to a staring end of the reaction channel,and an outlet port for releasing the fluid and an amplification productis connected to a terminal end of the reaction channel.
 23. Theapparatus of claim 22, wherein the heating unit is installed at a lowersurface of the substrate and comprises at least two heaters for heatingthe substrate to allow the substrate to have at least two differenttemperature areas.
 24. The apparatus of claim 22, wherein the heatingunit comprises a plurality of independently controllable heatersinstalled in parallel at a lower surface of the substrate to heat thesubstrate so that the substrate has at least two temperature areas. 25.The apparatus of claim 23, wherein the reaction channel alternately andrepeatedly passes through the temperature areas.
 26. The apparatus ofclaim 22, wherein the second inlet channel linearly extends from thestarting end of the reaction channel and the first inlet channel isformed perpendicularly to the second inlet channel.
 27. The apparatus ofclaim 22, further comprising: a third inlet channel formed inside thesubstrate, connected to the end of the reaction channel, and allowingintroduction of at least one selected from a negative control aqueoussolution and a positive control aqueous solution for quantitative assayinto the reaction channel; and a fourth inlet channel formed inside thesubstrate, connected to the end of the reaction channel, and allowingintroduction of a standard sample aqueous solution for quantitativeassay into the reaction channel.
 28. The apparatus of claim 27, whereinthe fourth inlet channel is connected to at least two standard sampleinlet channels for introduction of standard samples and a distilledwater inlet channel for diluting the standard samples to a predeterminedconcentration.
 29. The apparatus of claim 28, wherein the fourth inletchannel has a serpentine shape.
 30. The apparatus of claim 22, whereinthe apparatus comprises at least two first inlet channels and the firstinlet channels are correspondingly connected to at least two primerinlet channels for introduction of at least two different primers intothe first inlet channels.
 31. The apparatus of claim 30, wherein each ofthe first inlet channels has a serpentine shape.
 32. The apparatus ofclaim 22, wherein the first inlet channel is connected to a primer inletchannel for introduction of a primer into the first inlet channel and acommon component inlet channel for introduction of a common componentcomprising polymerase and dNTP into the first inlet channel.
 33. Theapparatus of claim 32, wherein a portion of a starting end side of thereaction channel has a serpentine shape.
 34. The apparatus of claim 15,wherein the reaction vessel comprises two serpentine primary reactionchannels and a single serpentine secondary reaction channel connected toa terminal end of each of the two primary reaction channels, and thefirst inlet channel and the second inlet channel are connected tostarting ends of the two primary reaction channels.
 35. The apparatus ofclaim 34, wherein the heating unit comprises a plurality ofindependently controllable heaters installed at a lower surface of thesubstrate to provide two temperature areas for each of the two primaryreaction channels and the single secondary reaction channel.
 36. Theapparatus of claim 15, wherein the reaction vessel is a reactionchamber, the first inlet channel and the second inlet channel areconnected to an end of the reaction chamber, and an outlet channel forreleasing the fluid and an amplification product is connected to theother end of the reaction chamber.
 37. The apparatus of claim 36,wherein the apparatus comprises a plurality of reaction chambers, firstinlet channels, second inlet channels, and outlet channels.
 38. Theapparatus of claim 36, wherein the heating unit comprises a singleheater installed at a lower surface of the substrate and heating theentire surface of the substrate to a predetermined temperature.